We report here a simple and experimentally tractable system for the infection and serial propagation of KSHV in human microvascular endothelium. Our optimized conditions allow for reproducible infection of 60 to 95% of the cells in a standard culture, although we note that substantial quantities of virions are required for a high density of infection even after such optimization. It is possible that serial passage of BCBL-1-derived virus on TIME cells may select for variants adapted to more efficient and high-titer replication in endothelia, and efforts to select such variants are now under way. If successful, such efforts could remove one remaining limitation of the present system, namely, that yields of infectious virus are modest. Although we present clear documentation of serial transmission of infectivity, the system in its present form is more suited to studies on the analytical rather than the preparative scale.
Our system differs substantially in design from that of Flore et al. (11
), so the two systems cannot be directly compared. Their system begins with primary (nonimmortalized) microvascular endothelium and scores for cell life span extension; ours begins with immortalized endothelium and scores for viral infection. Because the efficiency of latent infection is much higher in the TIME system, TIME cells are clearly preferable for studies of viral gene expression, viral stock preparation, and development of mutant viral strains. By contrast, the system of Flore et al. presents opportunities to explore possible roles of paracrine signaling that are not prominent features of the TIME system. Formally, our system is more analogous to that developed by Moses et al. (27
), in that both use life-extended microvascular endothelium as infection targets and report higher frequencies of infection. However, the human papillomavirus E6/E7-expressing endothelial cell line used in their studies grows slowly and erratically, and stable long-term passage of the cells has been problematic in many laboratories. Nonetheless, the results in both systems are in agreement that the default pathway for KSHV infection in endothelia is latency, just as in infection of primary B cells by Epstein-Barr virus but in contrast to the case of alphaherpesviruses like herpes simplex virus, which promptly enter the lytic cycle in most cultured lines.
Our results have three important implications for KSHV research. First, they open several aspects of the viral life cycle to experimental scrutiny. The availability of a simple system for de novo infection should facilitate the study of the early events of infection, such as envelope-receptor interactions, membrane fusion, and nuclear delivery. Related to this is the fact that the system should be readily adaptable to the development of assays for virus neutralization, either by patient antisera or by experimentally raised antiviral antibodies. Second, the ability to serially passage KSHV opens the door to the construction and analysis of mutant viruses in which individual KSHV genes have been disrupted by homologous recombination. Such systems have revolutionized our understanding of other herpesviruses and should now be possible for KSHV.
Finally, our results have implications for the study of KSHV latency in endothelia and its relationship to cellular growth and survival. Clearly, the system can be used to enumerate the viral genes expressed in endothelial latency, which will allow instructive comparison with the better-studied latency program in B cells. Because TIME cells are already immortalized, inferences about the effects of latency on cell growth and survival must be made with circumspection (the primary microvascular culture system of Ciufo et al. [5
] is superior for this sort of analysis). Certainly, infected TIME cells do not display the typical morphological changes described for malignantly transformed cells, nor do they lose contact inhibition or form foci in vitro. This accords well with the facts that KS spindle cells are diploid and that cultures derived from them also lack evidence of transformation, as they do not form colonies in soft agar or tumors in nude mice (10
). While we cannot exclude the possibility that the latency program may be an immortalizing stimulus in endothelia, we note that in TIME cells, in primary endothelial cells, and in authentic KS spindle cells explanted in culture (1
), latently infected cells are rapidly lost. We can envision two possible (and not mutually exclusive) explanations for this behavior. First, the plasmid maintenance function of LANA may be inefficient or nonfunctional in endothelial cells; if so, this would result in segregation of the viral episome with cell passage and the progressive accumulation of uninfected cells. Second, it is possible that the KSHV latency program may not lead to a sustained growth advantage under conditions employed in culture. Clearly, latently infected cells accumulate in KS tumors as they progress and therefore must have at least some competitive advantage over uninfected cells in vivo (31
). However, such an advantage need not be at the level of increased cell proliferation. If, for example, infected cells were more resistant to growth inhibition by inflammatory mediators released in the tumor microenvironment, a similar enrichment for infected cells could result in vivo but be undetectable in vitro. In either case, the net result would be that, at the level of the single cell, latent endothelial infection is of finite duration.
Recently, Martin et al. (25
) showed that treatment of patients with advanced AIDS with ganciclovir, a drug that blocks lytic but not latent KSHV infection, leads to a dramatic decrease in KS development. Thus, development of a KS lesion appears to require continuous lytic replication throughout the natural history of infection. Such a result is exactly what would be expected if the life span of an individual, latently infected spindle cell is not indefinite. Under such conditions, a tumor could form only if the rate of new latent infections generated by lytically produced virus exceeded the rate of disappearance of latently infected cells. Obviously, this clinical experiment can be interpreted in several other ways and by itself mandates no one model of tumorigenesis. However, at a minimum it can be said that nothing in the biology of KS requires that the latency program be transforming or even permanently life-extending, in the sense required to score in popular in vitro assays commonly used to define oncogenes. An important corollary of this is that searching for viral genes that score in such assays will not necessarily lead to identification of the critical determinants of KS pathogenesis.